PROJECT SUMMARY The multi-drug resistant bacterium S. maltophilia (Sm) is increasingly recognized as an important opportunistic and nosocomial pathogen. Pneumonia and blood stream infections are the most common manifestation of Sm infection, although Sm is also linked with CNS, eye, heart, skin, and urinary tract infections. Sm infection is also an independent risk factor for lung exacerbations in cystic fibrosis patients. Despite the emerging role of Sm in disease and the multi-drug resistance of Sm, our understanding of this Gram-negative bacterium's virulence is quite minimal. Thus, we embarked upon research aimed at developing methods to study Sm and identify factors that promote pathogenesis. After establishing a murine model for pneumonia, developing means for mutagenizing Sm, and documenting the importance of Sm's type II protein secretion system, we began to investigate the nature of Sm iron acquisition and the existence of a Sm siderophore. Past work from our lab and others had shown that siderophores are major facilitators of virulence in various other pathogens, including other agents of pneumonia and bloodstream infection. To begin, we determined that the genome of Sm strain K279a is predicted to encode a complete siderophore system, including a putative biosynthesis pathway, an outer membrane receptor for ferrisiderophore, and the other necessary import and export machinery. Compatible with these genomic data, we determined that K279a as well as other clinical isolates of Sm produce a siderophore-like activity when grown in a deferrated, chemically-defined medium. This activity was detected by both the Arnow assay which identifies catecholate structures and the Chrome Azurol S assay which detects high affinity iron chelators independently of structure. The secreted activity also had biological activity, as it rescued the growth of iron-starved Sm. When we mutated a gene in the putative biosynthesis locus of strain K279a, there was complete loss of both Arnow- and CAS-activity and bioactivity. Although the Sm biosynthesis locus had some similarity to that of enterobactin, a catecholate made by enteric bacteria, the Sm siderophore-containing supernatants were unable to rescue the growth of an enterobactin-utilizing indicator strain, and conversely iron-starved Sm could not use purified enterobactin. Together, our data suggest that Sm secretes a novel catecholate siderophore that is distinct from enterobactin. Thus, we hypothesize that the siderophore secreted by K279a is a virulence factor of Sm. To address our hypotheses, we propose to i) determine the identity (i.e., structure) of the Sm siderophore using both mass spectrometry and NMR analyses (Aim 1), and ii) test siderophore mutants in two different murine models of disease, the published pneumonia model and a new bacteremia model (Aim 2). The results of this grant will help establish a foundation for the nascent Sm field such that future efforts will be able to focus on characterizing systems and secreted factors that are most important. In addition to providing much-needed insight into Sm pathogenesis, the data obtained will define a new type of siderophore and thus may lead to new forms of disease treatment or prevention.